Smart polyhydroxyalkanoate nanobeads by protein based functionalization


Endotoxin free PHA nanobead production



Yüklə 250,95 Kb.
səhifə2/3
tarix11.01.2019
ölçüsü250,95 Kb.
#94734
1   2   3

6 Endotoxin free PHA nanobead production

Bacterial lipopolysaccharides (LPS) or endotoxins, also designated as pathogen associated molecular patterns (PAMPs) recognized by innate immune system are most potent identified microbial mediators implicated in the pathogenesis of sepsis and septic shock. LPS is the most prominent ‘alarm molecule’ sensed by the host’s early warning system of innate immunity presaging the threat of invasion by Gram-negative bacterial pathogens.95 Thus, presence of lipopolysaccharide (LPS) endotoxins in PHA nanobeads produced in Gram-negative bacteria make these in vivo naturally produced particles unsuitable for biomedical applications.96,97 The problem occurs because co-purification of pyrogenic outer LPS together with PHA granules cannot be avoided. In vitro approach on the other hand offers the possibility of endotoxin removal from PHA polymer. The concentration of endotoxins in PHA is greatly influenced by purification strategy and might vary from more than 104 EU/g to less than 1 EU/g.55,98 The methodology for endotoxin elimination depends on type of PHA (e.g., scl-PHA, mcl-PHA, presence of functional groups, etc.) and each results in different rates of polymer recovery.55,98 However, in vitro strategy remains hampered by the necessity of extensive and tedious purification methodology to achieve the levels in compliance with the endotoxin requirements for biomedical application according to the U.S. Food and Drug Administration (FDA). Generally, for products that directly or indirectly contact the cardiovascular system and lymphatic system the limit is 0.5 EU/mL or 20 EU/device, while for devices in contact with cerebrospinal fluid the limit is 0.06 EU/mL or 2.15 EU/device.99 All mentioned factors together with the bacteria growth conditions significantly influence the total cost of the production of endotoxin-free polymer. To get around this limitation, alternative sources of functionalized PHA granules free of LPS contamination are Gram-positive bacteria. They offer a platform for production of LPS free tailored beads due to the difference in the structure of their cell envelopes compared to Gram-negative bacteria.100 Even so, other PAMPs, such as lipoteichoic acid (LTA) and peptidoglycan (PG), found in Gram-positive bacterial pathogens are now appreciated to activate many of the same or similar host defense networks induced by LPS.95 Subsequently their presence in PHAs isolated from Gram-positive bacteria might have immunogenic activities similar to LPS.101 Among PAMPs, LTA predominate in the Bacillus, whereas actinomycete bacteria typically synthesize lipoglycans.102 Importantly, certain Gram-positive PHA producing strains (e.g. Bacillus circulans, Bacillus polymyxa) lack both, LTA and lipoglycans.103 Clostridium and Staphylococcus citreus were reported to lack LTA and may be considered for recombinant PHA production.104 Hence, emerging area to be investigated are the mechanisms triggered by PAMPs of Gram-positive PHA producing bacteria regarding mammalian immune system. Remarkably, Gram-positive genera Corynebacterium, Nocardia and Rhodococcus are the only wild-type bacteria, which naturally synthesize the commercially important copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), from simple carbon sources such as glucose.105,106 The genus Bacillus, in common with many other PHA-accumulating Gram-positive bacteria, accumulates co-polymers of 3HB when grown on different substrates.98 For instance, copolymers of P(3HB-co-3HV) are accumulated when the cultures are fed with odd-chain-length n-alkanoic acids such as propionic acid, valeric acid and heptanoic acid.107

The generally-regarded-as-safe (GRAS) bacterium Lactococcus lactis has been genetically engineered to produce PHA beads. Unfortunately this recombinant strain did not show feasibility for commercial-scale production, since the beads were both smaller in size and contributed less PHA per CDW (6%) then other PHA producing bacteria.29 Therefore, this platform was designated for added value medical product synthesis (e.g., vaccine development) instead the large scale production.25 The improvement of the yield would likely require re-engineering metabolic flux to push carbon utilization away from lactate production and toward the PHA biosynthesis pathway.29 Interestingly, the platform based on PHA functionalized granules was used to develop a PhaP-based system for endotoxin removal from protein solution. An endotoxin receptor protein was fused with R. eutropha phasin, in vitro attached to PHB beads and used to remove LPS from the solution.108



7 Functionalized PHA nanobead in vivo performance, cytotoxicity and biocompatibility

Numerous in vivo studies have clearly demonstrated that endotoxin and bacterial protein free PHAs provoke mild host reactions in different animal models,96 which is not surprising when considering the fact that [R]-3-hydroxybutyric acid is a normal blood constituent109 and is found in the cell envelope of eukaryotes.110 In vitro based approaches have focused on enhancing growth of different eukaryotic cell lines using Arginyl-glycyl-aspartic acid (RGD) tailored PHA in form of a scaffold. As such, it showed excellent in vitro performance on supporting and promoting neural stem cell, human bone marrow mesenchymal stem cell, fibroblasts adhesion and growth.111– 113 PhaP-RGD fusion immobilization allowed evading tedious cross-linking processes and chemical immobilization that easily damage the biological activity of attached protein. New approaches based on nanoparticulate carriers with targeting capability for imaging and drug delivery to cancer cells are slowly replacing longstanding concepts. With this aim, posterior to synthesis of loaded PHA particles, surface modification was performed via hydrophobic interaction between particle surface and growing PHA chain from PhaC enzyme fusion with RGD that stabilized core-shell structure.31 However, little attention was placed on endotoxin removal and scaffold performance in vivo. Alternatively, the PHA micelles synthesis was performed in vitro by mixing PhaC-RGD and 3HB-CoA and therefore avoiding the incorporation of endotoxins.66

Bacterial polyester inclusions have been also engineered to display fusion protein of PhaC and the components involved in immune response to the infectious agent and used as a vaccine delivery system.19 Remarkably, particle-based carriers very closely mimic the physiochemical characteristics of natural pathogens, enhancing particle-displayed protein delivery to the immune system.114–116 However, very few in vivo studies address essential issue of immunogenicity of soluble and PHA granule bound GAPs, considering that the main objective when using biomaterials and nanocariers is to generate the most appropriate beneficial cellular or tissue response without eliciting any undesirable local or systemic effects in the recipient of the therapy. As the immune response and repair functions in the body are exceptionally complex, the biocompatibility of a material should not be described in relation to a single cell type or tissue. Nevertheless, it is essential to consider in vitro and in vivo cellular behavior for further comprehensive biocompatibility evaluation of biopolymers.

Several studies report no toxic nor pyrogenic effect of wild type or functionalized non endotoxin free PHA beads in mice,19 which suggests that due to the profound differences between mice and human immune systems another animal model should be considered for these type of studies.117 Given the breadth of these functional differences, the discrepancies surely limit the usefulness of mouse models in mentioned studies and as such should be taken into account when choosing preclinical animal models.118 The results of the study comparing immune response of PHA-beads for vaccine application produced in L. lactis and E. coil support this hypothesis since no higher inflammation was spotted for E. coli produced particles.26,29 However, this might be due to the PAMPs, present in both Gram-positive and Gram-negative bacteria that induce similar immune reaction. In addition, overall impact of functionalized PHA nanobeads on eukaryotic organism including levels of ketone bodies and other possible secondary effects are unknown. In vivo tracking of PHA nanocarriers might give insight into environmentally-triggered structural changes of nanoparticles and provide additional information about their localization and pathway.



8 PHA in mammalian cells

In a very different context, complexed PHA (cPHA) were discovered representing different type of PHA structures. Unlike bacterial PHA that play a major role in carbon and energy storing, these cPHA found in mammalian cells are assumed to be involved in regulation of various cell functions through modification of target molecules.119 Complex of cPHA with Ca2+ and inorganic polyphosphate is involved in formation of ion-conducting channels in mitochondrial membranes.120 Furthermore, cPHA can interact with membrane proteins through hydrophobic and perhaps covalent interactions.121,122 It has been suggested that in case of protein channels these interactions might play an important role in regulation of channel function and selectivity.123 Previous studies indicate that cPHA can be found in various subcellular compartments of the eukaryotic organisms124 as well as associated with specific proteins.125,126 Although, these structures are still not profoundly explored and are in very early stage of investigation, they definitely offer great possibility for functionalization and exploitation. Additionally, they might give the critical piece of information on PHA metabolism, their uptake and pathway inside the eukaryotic cell essential when dealing with functionalized PHA nanobeads designed for biomedical application.



9 Bacterial polyesters and their synthetic competitors

Besides natural polyesters such as PHA, several synthetic polyesters have attracted considerable attention as materials for biomedical purposes due to their attractive properties (e.g., biocompatibility and biodegradability). Currently majority of synthetic polyesters systems used in medicine are based on poly(lactic acid) PLA, poly(glycolic acid) PGA and their copolymer poly(lactic-co-glycolic acid) PLGA. This is mainly due to their well described formulations and methods for production, as well as their low toxicity and immunogenicity. Even though such polyesters have been extensively used for resorbable sutures, bone implants, screws and others,127 only small number of commercially available products are designed for nanoparticle based drug delivery.128 Nevertheless, synthetic polyesters such as PLGA have been profoundly tested for this application (reviewed in 128,129).

Synthetic polyesters are considered promising candidates for development of the nanoparticle delivery systems to release, target, uptake, retain, activate and localize the drugs at the right time, place and dose.130 Although natural and synthetic polyesters share many common properties (e.g., biocompatibility and biodegradability), due to their specific characteristic one or the other might be more suitable dependently on the application. The main characteristic of synthetic and natural polyesters, significant for nanoparticle production and drug delivery systems are outlined in Table 3. Degradation of both, synthetic and natural polyesters, results in biologically compatible and metabolizable moieties. However, their degradation rates and patterns differ considerably. Thereby, synthetic polyesters are suitable for sustained release due to their slow degradation rates. Importantly, in the case of natural polyesters the drug release kinetics can be more easily controlled via conventionally engineering the PHA matrix parameters to reach desired degradation rates. For instance, scl-PHAs are crystalline and hydrophobic, but many pores are formed on the surface and the drugs are released quickly without any polymer degradation. Mcl-PHA copolymers on the other hand, have low melting point and low crystallinity, therefore they are more suitable for drug delivery.

PLGA found many applications in biomedical field, such as treatment of cancer, inflammation diseases, cerebral diseases, cardio-vascular disease as well as in regenerative medicine, infection treatment, vaccination and many others.128,133 They were also used for diagnostic purposes for magnetic resonance, cancer-targeted imaging 137,138 and as ultrasound contrast agent.139 Similarly, the good performance of PHAs for variety of biomedical applications has been proven (Tab. 1). Nevertheless, the main advantage of synthetic PLGA over natural PHAs is its FDA approval as drug delivery platform and lower production costs. Currently, the only FDA approved PHA is poly(4-hydroxybutirate) P(4HB) for suture application, which might open the possibility for other PHAs to be tested and enter the investigations for FDA approval. This would significantly influence the development of PHA based drug delivery systems and enhance their application.



At present, due to its large availability on the market and its relatively low price, PLA shows one of the highest potential among polyesters, particularly for packaging and medical applications. For instance, Cargill has developed processes that use corn and other feedstock to produce different PLA grades (NatureWorks).140 In this company, the actual production is estimated to be 140,000 tons/year. Presently, it is the highest and worldwide production of biodegradable polyester. Its price is lower than 2 €/kg.141 Although, the cost of production of PHAs is still quite high (3–5 €/kg), current advances in fermentation, extraction and purification technology as well as the development of superior bacterial strains are likely to lower the price of PHAs, close to that of other biodegradable polymers such as polylactide and aliphatic polyesters.142

10 Conclusions

Engineering biomaterial nanobeads has attracted much attention of the research community. Ongoing efforts to push the boundaries are reflected in the design of wide range of nanostructured bacterial materials for innovative medicines.1 Apart from PHA, biologically produced nanoparticles are highly diverse and omnipresent in prokaryotic (magnetosomes, storage paricles, etc.), but also in eukaryotic (e.g., exosomes, lipoproteins, etc.) systems giving the ground to the further development of bionanothechnology.11 Smart PHA nanoparticles described in this review provide grounds on how these bacterial polymers, traditionally considered for industrial or conventional clinical applications, are progressively entering the most innovative biomedical fields as promising and highly flexible materials. The fact that PHA can be produced from inexpensive waste carbon sources enhanced commercial interest in these polymers. On the other hand, interest in functionalized PHA nanobead technology has been hampered by existing legislation in terms of endotoxin concentration allowed for biomedical application.99 Importantly, these technical hurdles were successfully surmounted following in vitro approach or using certain Gram-positive strains for in vivo functionalized bead assembly. Nevertheless, up-to-date PHAs are produced on the large-scale exclusively using Gram-negative bacteria.4 For simplicity and cost control the goal is to adapt the approach to a system in which maximal covering of PHA granule surface with recombinant protein is achieved. Different module swapping strategies and fine tuning were proven effective to reach this goal.8 To meet the challenges new tendencies suggest multi-functionality. The concept behind multi-functional beads would allow the design of variety of biomedical systems with unique advantage of adaptability and subsequently responding to current trends of biomedicine. PHA nanoparticles allow multifunctional tuning due to the possibility of the use of variety of GAPs, as well as their both N- and C-terminal domains, to immobilize diverse proteins simultaneously. Nevertheless, many nanotoxicological test on their safety have to be performed before they can overtake the current stage of synthetic polyesters. Aside from FDA approval for biomedical applications, the production costs should be reduced. The big challenges that PHA industry has to overcome132 to lead to PHA nanobeads successfully commercialization are: i) reduction of production costs; ii) construction of functional PHA production strains to precisely control the structure of PHA molecules increasing the consistency of structure and properties to reach the level of competitor synthetic polymers; iii) reach the simplicity of synthetic polymer processing; iv) use of alternative renewable sources for production to avoid use of expensive glucose; v) development of high value added applications.

References

1. Rodríguez-Carmona E, Villaverde A. Nanostructured bacterial materials for innovative medicines. Trends Microbiol 2010;18:423–430.

2. Draper JL, Rehm BH. Engineering bacteria to manufacture functionalized polyester beads. Bioengineered 2012;3:203–208.

3. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev 2003;55:329–347.

4. Chen GQ. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem Soc Rev 2009;38:2434–2446.

5. Tortajada M, da Silva LF, Prieto MA. Second-generation functionalized medium-chain-length polyhydroxyalkanoates: the gateway to high-value bioplastic applications. Int Microbiol 2013;16:1–15.

6. Dinjaski N, Fernández-Gutiérrez M, Selvam S, Parra-Ruiz FJ, Lehman SM, San Román J, García E, García JL, García AJ, Prieto MA. PHACOS, a functionalized bacterial polyester with bactericidal activity against methicillin-resistant Staphylococcus aureus. Biomaterials 2014;35:14–24.

7. Moldes C, García P, García JL, Prieto MA. In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF. Appl Environ Microbiol 2004;70:3205–3212.

8. Dinjaski N, Prieto MA. Swapping of phasin modules to optimize the in vivo immobilization of proteins to medium-chain-length polyhydroxyalkanoate granules in Pseudomonas putida. Biomacromolecules 2013;14:3285–3293.

9. Galán B, Dinjaski N, Maestro B, de Eugenio LI, Escapa IF, Sanz JM, García JL, Prieto, MA. Nucleoid-associated PhaF phasin drives intracellular location and segregation of polyhydroxyalkanoate granules in Pseudomonas putida KT2442. Mol Microbiol 2011;79:402–418.

10. Jendrossek D, Pfeiffer D. New Insights in Formation of Polyhydroxyalkanoate (PHA) granules (Carbonosomes) and Novel Functions of poly(3-hydroxybutyrate) (PHB). Environ Microbiol 2013;doi: 10.1111/1462-2920.12356.

11. Stanley S. Biological nanoparticles and their influence on organisms. Current Opinion in Biotechnology 2014;28:69–74.

12. Jendrossek D. Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes). J Bacteriol 2009;191:3195–3202.

13. Lewis JG, Rehm BHJ. ZZ polyester beads: an efficient and simple method for purifying IgG from mouse hybridoma supernatants. Immunol Methods 2009;346:71–74.

14. Peters V, Rehm BH. In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase. Appl Environ Microbiol 2006;72:1777–1783.

15. Chen SY, Chien YW, Chao YP. In vivo immobilization of d-hydantoinase in Escherichia coli. J Biosci Bioeng 2014;pii:S1389-1723(13)00478-7.

16. Moldes C, Farinós GP, de Eugenio LI, García P, García JL, Ortego F, Hernández-Crespo P, Castañera P, Prieto MA. New tool for spreading proteins to the environment: Cry1Ab toxin immobilized to bioplastics. Appl Microbiol Biotechnol 2006;72:88–93.

17. Blatchford PA, Scott C, French N, Rehm BH. Immobilization of organophosphohydrolase OpdA from Agrobacterium radiobacter by overproduction at the surface of polyester inclusions inside engineered Escherichia coli. Biotechnol Bioeng 2012;109:1101–1108.

18. Bäckström BT, Brockelbank JA, Rehm BHA. Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein. BMC Biotechnol 2007;7:3.

19. Parlane NA, Wedlock DN, Buddle BM, Rehm BH. Bacterial polyester inclusions engineered to display vaccine candidate antigens for use as a novel class of safe and efficient vaccine delivery agents. Appl Environ Microbiol 2009;75:7739–7744.

20. Grage K, Jahns AC, Parlane N, Palanisamy R, Rasiah IA, Atwood JA, Rehm BH. Bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-/micro-beads in biotechnological and biomedical applications. Biomacromolecules 2009;10:660–669.

21. Atwood JA, Rehm BH. Protein engineering towards biotechnological production of bifunctional polyester beads. Biotechnol Lett 2009;31:131–137.

22. Lee SJ, Park JP, Park TJ, Lee SY, Lee S, Park JK. Selective immobilization of fusion proteins on poly(hydroxyalkanoate) microbeads. Anal Chem 2005;77:5755–5759.

23. Chen S, Parlane NA, Lee J, Wedlock DN, Buddle BM, Rehm BH. New skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant Escherichia coli. Appl Environ Microbiol. 2014;80:2526–2535.

24. Grage K, Rehm BH. In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner. Bioconjug Chem 2008;19:254–262.

25. Mifune J, Grage K, Rehm BH. Production of functionalized biopolyester granules by recombinant Lactococcus lactis. Appl Environ Microbiol 2009;75:4668–4675.

26. Parlane NA, Rehm BH, Wedlock DN, Buddle BM. Novel particulate vaccines utilizing polyester nanoparticles (bio-beads) for protection against Mycobacterium bovis infection-A review. Vet Immunol Immunopathol 2014;158:8–13.

27. Parlane NA, Grage K, Mifune J, Basaraba RJ, Wedlock DN, Rehm BH, Buddle BM. Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis. Clin Vaccine Immunol 2012;19:37–44.

28. Rice-Ficht AC, Arenas-Gamboa AM, Kahl-McDonagh MM, Ficht TA. Polymeric particles in vaccine delivery. Curr Opin Microbiol 2010;13:106–112.

29. Parlane NA, Grage K, Lee JW, Buddle BM, Denis M, Rehm BH. Production of a particulate hepatitis C vaccine candidate by an engineered Lactococcus lactis strain. Appl Environ Microbiol 2011;77:8516–8522.

30. Yao YC, Zhan XY, Zhang J, Zou XH, Wang ZH, Xiong YC, Chen J, Chen GQ. A specific drug targeting system based on polyhydroxyalkanoate granule binding protein PhaP fused with targeted cell ligands. Biomaterials 2008;29:4823–4830.

31. Lee J, Jung SG, Park CS, Kim HY, Batt CA, Kim YR. Tumor-specific hybrid polyhydroxybutyrate nanoparticle: surface modification of nanoparticle by enzymatically synthesized functional block copolymer. Bioorg Med Chem Lett 2011;21:2941–2944.

32. Xiong YC, Yao YC, Zhan XY, Chen GQ. Application of polyhydroxyalkanoates nanoparticles as intracellular sustained drug-release vectors. J Biomater Sci Polym Ed 2010;21:127–140.

33. Kassab AC, Xu K, Denkbaş EB, Dou Y, Zhao S, Pişkin E. Rifampicin carrying polyhydroxybutyrate microspheres as a potential chemoembolization agent. J Biomater Sci Polym Ed 1997;8:947–961.

34. Bayram C, Denkbas EB, Kiliçay E, Hazer B, Çakmak HB, Noda I. Preparation and Characterization of Triamcinolone Acetonide-loaded Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHx) Microspheres. Journal of Bioactive and Compatible Polymers 2008;23:334–347.

35. Bissery MC, Valeriote F, Thies C. Fate and effect of CCNU-loaded microspheres made of poly(d,l)lactide (PLA) or poly-β-hydroxybutyrate (PHB) in mice. Proc Int Symp Controlled Release Bioact Mater 1985;12:181–182.

36. Heathman TR, Webb WR, Han J, Dan Z, Chen GQ, Forsyth NR, El Haj AJ, Zhang ZR, Sun X. Controlled production of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) nanoparticles for targeted and sustained drug delivery. J Pharm Sci 2014;103:2498–2508.

37. Murueva AV, Shershneva AM, Shishatskaya EI, Volova TG. The use of polymeric microcarriers loaded with anti-inflammatory substances in the therapy of experimental skin wounds. Bull Exp Biol Med 2014;157:597–602.

38. Kılıçay E, Demirbilek M, Türk M, Güven E, Hazer B, Denkbas EB. Preparation and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHX) based nanoparticles for targeted cancer therapy. Eur J Pharm Sci 2011;44:310–320.

39. Dong CL, Webb WR, Peng Q, Tang JZ, Forsyth NR, Chen GQ, El Haj AJ. Sustained PDGF-BB release from PHBHHx loaded nanoparticles in 3D hydrogel/stem cell model. J Biomed Mater Res A. 2014;doi: 10.1002/jbm.a.35149.

40. Wu LP, Wang D, Parhamifar L, Hall A, Chen GQ, Moghimi SM. Poly(3-hydroxybutyrate-co-R-3-hydroxyhexanoate) nanoparticles with polyethylenimine coat as simple, safe, and versatile vehicles for cell targeting: population characteristics, cell uptake, and intracellular trafficking. Adv Healthc Mater 2014;3:817–824.

41. Peters V, Rehm BH. In vivo monitoring of PHA granule formation using GFP-labeled PHA synthases. FEMS Microbiol Lett 2005;248:93–100.

42. Peters V, Becher D, Rehm BH. The inherent property of polyhydroxyalkanoate synthase to form spherical PHA granules at the cell poles: the core region is required for polar localization. J Biotechnol 2007;132:238–245.

43. Jahns AC, Haverkamp RG, Rehm BH. Multifunctional inorganic-binding beads self-assembled inside engineered bacteria. Bioconjug Chem 2008;19:2072–2080.

44. Jahns AC, Rehm BH. Tolerance of the Ralstonia eutropha class I polyhydroxyalkanoate synthase for translational fusions to its C terminus reveals a new mode of functional display. Appl Environ Microbiol 2009;75:5461–5466.

45. Brockelbank JA, Peters V, Rehm BH. Recombinant Escherichia coli strain produces a ZZ domain displaying biopolyester granules suitable for immunoglobulin G purification. Appl Environ Microbiol 2006;72:7394–7397.

46. Peters V, Rehm BH. Protein engineering of streptavidin for in vivo assembly of streptavidin beads. J Biotechnol 2008;134:266–274.

47. Banki MR, Gerngross TU, Wood DW. Novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Science 2005;14:1387–1395.

48. Barnard GC, McCool JD, Wood DW, Gerngross TU. Integrated recombinant protein expression and purification platform based on Ralstonia eutropha. Appl Environ Microbiol 2005;71:5735–5742.

49. Wang Z, Wu H, Chen J, Zhang J, Yao Y, Chen GQ. A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles. Lab Chip 2008;8:1957–1962.

50. Rasiah IA, Rehm BH. One-step production of immobilized alpha-amylase in recombinant Escherichia coli. Appl Environ Microbiol 2009;75:2012–2016.

51. Mullaney JA, Rehm BH. Design of a single-chain multi-enzyme fusion protein establishing the polyhydroxybutyrate biosynthesis pathway. J Biotechnol 2010;147:31–36.

52. Li Y. Self-cleaving fusion tags for recombinant protein production. Biotechnol Lett 2011;33:869–881.

53. Leong YK, Show PL, Ooi CW, Ling TC, Lan JC. Current trends in polyhydroxyalkanoates (PHAs) biosynthesis: insights from the recombinant Escherichia coli. J Biotechnol 2014;180:52–65.

54. Rehm BH. Biogenesis of microbial polyhydroxyalkanoate granules: a platform technology for the production of tailor-made bioparticles. Curr Issues Mol Biol 2007;9:41–62.

55. Furrer P, Panke S, Zinn M. Efficient recovery of low endotoxin medium-chain-length poly([R]-3-hydroxyalkanoate) from bacterial biomass. J Microbiol Methods 2007;69:206–213.

56. Rehm BH. Bacterial polymers: biosynthesis, modifications and applications. Nat Rev Microbiol 2010;8:578–592.

57. Nogales J, Palsson BØ, Thiele I. A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory. BMC Syst Biol 2008;2:79.doi:10.1186/1752-0509-2-79.

58. Maestro B, Galán B, Alfonso C, Rivas G, Prieto MA, Sanz JM. A new family of intrinsically disordered proteins: structural characterization of the major phasin PhaF from Pseudomonas putida KT2440. PLoS One 2013;8,e56904.

59. Ihssen J, Magnani D, Thöny-Meyer L, Ren Q. Use of extracellular medium chain length polyhydroxyalkanoate depolymerase for targeted binding of proteins to artificial poly[(3-hydroxyoctanoate)-co-(3-hydroxyhexanoate)] granules. Biomacromolecules 2009;10:1854–1864.

60. Park TJ, Yoo SM, Keum KC, Lee SY. Microarray of DNA-protein complexes on poly-3-hydroxybutyrate surface for pathogen detection. Anal Bioanal Chem 2009;393:1639–1647.

61. Muniasamy G, Pérez-Guevara F. Use of SNAREs for the immobilization of poly-3-hydroxyalkanoate polymerase type II of Pseudomonas putida CA-3 in secretory vesicles of Saccharomyces cerevisiae ATCC 9763. J Biotechnol 2014;172:77–79.

62. Hooks DO, Blatchford PA, Rehm BH. Bioengineering of bacterial polymer inclusions catalyzing the synthesis of N-acetylneuraminic acid. Appl Environ Microbiol 2013;79:3116–3121.

63. McCool GJ, Cannon MC. PhaC and PhaR are required for polyhydroxyalkanoic acid synthase activity in Bacillus megaterium. J Bacteriol 2001;183:4235–4243.

64. Molino NM, Wang S-W. Caged protein nanoparticles for drug delivery. Current Opinion in Biotechnology 2014;28:75–82.

65. Steinmann B, Christmann A, Heiseler T, Fritz J, Kolmar H. In vivo enzyme immobilization by inclusion body display. Appl Environ Microbiol 2010;76:5563–5569.

66. Kim HN, Lee J, Kim HY, Kim YR. Enzymatic synthesis of a drug delivery system based on polyhydroxyalkanoate-protein block copolymers. Chem Commun (Camb) 2009;46:7104–7106.

67. Grage K, Peters V, Rehm BH. Recombinant protein production by in vivo polymer inclusion display. Appl Environ Microbiol 2011;77:6706–6709.

68. Geng Y, Wang S, Qi Q. Expression of active recombinant human tissue-type plasminogen activator by using in vivo polyhydroxybutyrate granule display. Appl Environ Microbiol 2010;76:7226–7230.

69. Chen GQ, Whang ZG. 2011 A method and kit for purification of recombinant proteins using self-cleaving protein intein. PTC/CN2008/001006.

70. Kim DY, Kim HC, Kim SY, Rhee YH. Molecular characterization of extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase genes from Pseudomonas alcaligenes strains. J Microbiol 2005;43:285–294.

71. Prieto MA, de Eugenio LI, Galán B, Luengo JM, Witholt B. in: “Pseudomonas: a Model System in Biology. Pseudomonas vol. V”, J.L. Ramos, A. Filloux, Springer. 2007.

72. Ouyang SP, Luo RC, Chen SS, Liu Q, Chung A, Wu Q, Chen GQ. Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442. Biomacromolecules 2007;8:2504–2511.

73. Qi Q, Steinbüchel A, Rehm BH. Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid. FEMS Microbiol Lett. 1998;167:89 FEMS Microbiol Lett. 1998 Oct 1;167(1):89–94.

74. Lu XY, Wu Q, Zhang WJ, Zhang G, Chen GQ. Molecular cloning of polyhydroxyalkanoate synthesis operon from Aeromonas hydrophila and its expression in Escherichia coli. Biotechnol Prog 2004;20:1332–1336.

75. Tsuge T, Taguchi K, Seiichi T, Doi Y. Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation. Int J Biol Macromol 2003;31:195–205.

76. Nomura CT, Taguchi K, Gan Z, Kuwabara K, Tanaka T, Takase K, Doi Y. Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109. Appl Environ Microbiol 2005;71:4297–4306.

77. Langenbach S, Rehm BH, Steinbüchel A. Functional expression of the PHA synthase gene phaC1 from Pseudomonas aeruginosa in Escherichia coli results in poly(3-hydroxyalkanoate) synthesis. FEMS Microbiol Lett 1997;150:303–309.

78. Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C, Porro D, Ferrer P, Tutino ML, Mattanovich D, Villaverde A. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 2008;7:11.

79. Poblete-Castro I, Becker J, Dohnt K, dos Santos VM, Wittmann C. Industrial biotechnology of Pseudomonas putida and related species. Appl Microbiol Biotechnol 2012;93:2279–2290.

80. Poblete-Castro I, Escapa IF, Jäger C, Puchalka J, Lam CM, Schomburg D, Prieto MA, Martins dos Santos VA. The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach. Microb Cell Fact 2012;11:34.doi:10.1186/1475-2859-11-34.

81. Poblete-Castro I, Binger D, Rodrigues A, Becker J, Martins Dos Santos VA, Wittmann C. In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates. C Metab Eng 2013;15:113–123.

82. Follonier S, Escapa IF, Fonseca PM, Henes B, Panke S, Zinn M, Prieto MA. New insights on the reorganization of gene transcription in Pseudomonas putida KT2440 at elevated pressure. Microb Cell Fact 2013;12:30.doi:10.1186/1475-2859-12-30.

83. Escapa IF, García JL, Bühler B, Blank LM, Prieto MA. The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida. Environ Microbiol 2012;14:1049–1063.

84. Silva-Rocha R, Martínez-García E, Calles B, Chavarría M, Arce-Rodríguez A, de Las Heras A, Páez-Espino AD, Durante-Rodríguez G, Kim J, Nikel PI, Platero R, de Lorenzo V. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res 2013;41:D666–D675.

85. Martínez V, García P, García JL, Prieto MA. Controlled autolysis facilitates the polyhydroxyalkanoate recovery in Pseudomonas putida KT2440. Microbial Biotechnology 2011;4:533–547.

86. Rehm BH, Steinbüchel A. Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 1999;25:3–19.

87. Pfeiffer D, Wahl A, Jendrossek D. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16. Mol Microbiol 2011;82:936–951.

88. Pfeiffer D, Jendrossek D. PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha. Appl Environ Microbiol 2014;80:555–563.

89. Stubbe J, Tian J. Polyhydroxyalkanoate (PHA) hemeostasis: the role of PHA synthase. Nat Prod Rep 2003;20:445–457.

90. Pfeiffer D, Jendrossek D. Interaction between poly(3-hydroxybutyrate) granule-associated proteins as revealed by two-hybrid analysis and identification of a new phasin in Ralstonia eutropha H16. Microbiology 2011;157:2795–2807.

91. Dennis D, Sein V, Martinez E, Augustine B. PhaP is involved in the formation of a network on the surface of polyhydroxyalkanoate inclusions in Cupriavidus necator H16. J Bacteriol 2008;190:555–563.

92. Prieto MA, Buehler B, Jung K, Witholt B, Kessler B. PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes. Bacteriol 1999;181:858–868.

93. Wahl A, Schuth N, Pfeiffer D, Nussberger S, Jendrossek D. PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha. BMC Microbiol 2012;12:262.

94. Neumann L, Spinozzi F, Sinibaldi R, Rustichelli F, Pötter M, Steinbüchel A. Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules. J Bacteriol 2008;190:2911–2919.

95. Opal SM. Endotoxemia and Endotoxin Shock: Disease, Diagnosis and Therapy. Contrib Nephrol. Ronco C, Piccinni P, Rosner MH (eds). Basel, Karger, 2010, vol 167, pp 14–24.

96. Chen GQ, Wu Q. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 2005;26:6565–6578.

97. Valappil SP, Misra SK, Boccaccini AR, Roy I. Biomedical applications of polyhydroxyalkanoates: an overview of animal testing and in vivo responses. Expert Rev Med Devices 2006;3:853–868.

98. Lee SY, Choi Ji, Han K, Song JY. Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria. Appl Environ Microbiol 1999;65:2762–2764.

99. FDA. Guideline on validation of the Limulus amebocyte lysate test as an end-product endotoxin test for human an animal parenteral drugs, biological products, and medical devices. In: U.S. Department of Health and Human Services FaDA, editor. Rockville, MD1987. http://www.gmpua.com/Validation/Method/LAL/FDAGuidelineForTheValidationA.pdf.

100. Valappil SP, Boccaccini AR, Bucke C, Roy I. Polyhydroxyalkanoates in Gram-positive bacteria: insights from the genera Bacillus and Streptomyces. Antonie Van Leeuwenhoek 2007;91:1–17.

101. Declue AE, Johnson PJ, Day JL, Amorim JR, Honaker AR. Pathogen associated molecular pattern motifs from Gram-positive and Gram-negative bacteria induce different inflammatory mediator profiles in equine blood. Vet J 2012;192:455–460.

102. Sutcliffe IC. The lipoteichoic acids and lipoglycans of gram-positive bacteria: a chemotaxonomic perspective. Syst Appl Microbiol 1994;17:467–480.

103. Iwasaki H, Shimada A, Yokoyama K, Ito E. Structure and glycosylation of lipoteichoic acids in Bacillus strains. J Bacteriol 1989;171:424–429.

104. Sutcliffe IC, Shaw N. Atypical lipoteichoic acids of gram-positive bacteria. J Bacteriol 1991;173:7065–7069.

105. Haywood GW, Anderson AJ, Williams DR, Dawes EA, Ewing DF. Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int J Biol Macromol 1991;13:83–88.

106. Alvarez HM, Kalscheuer R, Steinbüchel A. Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126. Appl Microbiol Biotechnol 2000;54:218–223.

107. Chen GQ, König KH, Lafferty RM. Production of poly-D(-)-3-hydroxybutyrate and poly-D(-)-3-hydroxyvalerate by strains of Alcaligenes latus. Antonie Van Leeuwenhoek 1991;60:61–66.

108. Li J, Shang G, You M, Peng S, Wang Z, Wu H, Chen GQ. Endotoxin removing method based on lipopolysaccharide binding protein and polyhydroxyalkanoate binding protein PhaP. Biomacromolecules 2011;12:602–608.

109. Wiggam MI, O'Kane MJ, Harper R, Atkinson AB, Hadden DR, Trimble ER, et al. Treatment of diabetic ketoacidosis using normalization of blood 3-hydroxybutyrate concentration as the endpoint of emergency management. A randomized controlled study. Diabetes Care 1997;20:1347–1352.

110. Reusch RN. Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes. Biochemistry (Mosc) 2000;65:280–295.

111. You M, Peng G, Li J, Ma P, Wang Z, Shu W, Peng S, Chen GQ. Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide. Biomaterials 2011;32:2305–2313.

112. Xie H, Li J, Li L, Dong Y, Chen GQ, Chen KC. Enhanced proliferation and differentiation of neural stem cells grown on PHA films coated with recombinant fusion proteins. Acta Biomater 2013;9:7845–7854.

113. Dong Y, Li P, Chen CB, Wang ZH, Ma P, Chen GQ. The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD. Biomaterials 2010;31:8921–8930.

114. Rosenthal JA, Chen L, Baker JL, Putnam D, DeLisa MP. Pathogen-like particles: biomimetic vaccine carriers engineered at the nanoscale. Current Opinion in Biotechnology 2014;28:51–58.

115. Newman KD, Samuel J, Kwon G. Ovalbumin peptide encapsulated in poly(d,l lactic-co-glycolic acid) microspheres is capable of inducing a T helper type 1 immune response. J Control Release 1998;54:49–59.

116. Singh M, Chakrapani A, O'Hagan D. Nanoparticles and microparticles as vaccine-delivery systems. Expert Rev Vaccines 2007;6:797–808.

117. Mestas J, Hughes CCW. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731–2738.

118. Roep BO, Atkinson M. Animal models have little to teach us about Type 1 diabetes: 1. In support of this proposal. Diabetologia 2004;47:1650–1656.

119. Elustondo P, Zakharian E, Pavlov E. Identification of the polyhydroxybutyrate granules in mammalian cultured cells. Chem Biodivers 2012;9:2597–2604.

120. Pavlov E, Zakharian E, Bladen C, Diao CT, Grimbly C, Reusch RN, French RJ. A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction. Biophys J 2005;88:2614–2625.

121. Reusch RN. Low molecular weight complexed poly(3-hydroxybutyrate): a dynamic and versatile molecule in vivo. Can J Microbiol 1995;41:50–54.

122. Reusch RN, Shabalin O, Crumbaugh A, Wagner R, Schröder O, Wurm R. Posttranslational modification of E. coli histone-like protein H-NS and bovine histones by short-chain poly-(R)-3-hydroxybutyrate (cPHB). FEBS Lett 2002;527:319–322.

123. Negoda A, Xian M, Reusch RN. Insight into the selectivity and gating functions of Streptomyces lividans KcsA. Proc Natl Acad Sci U S A. 2007;104:4342–4346.

124. Reusch RN. Poly-beta-hydroxybutyrate/calcium polyphosphate complexes in eukaryotic membranes. Proc Soc Exp Biol Med 1989;191:377–381.

125. Seebach D, Brunner A, Bürger HM, Schneider J, Reusch RN. Isolation and 1H-NMR spectroscopic identification of poly(3-hydroxybutanoate) from prokaryotic and eukaryotic organisms. Determination of the absolute configuration (R) of the monomeric unit 3-hydroxybutanoic acid from Escherichia coli and spinach. Eur J Biochem 1994;224:317–328.

126. Zakharian E, Thyagarajan B, French RJ, Pavlov E, Rohacs T. Inorganic polyphosphate modulates TRPM8 channels. PLoS One 2009;4(4):e5404. doi: 10.1371/journal.pone.0005404.

127. Gomes ME, Reis RL. Biodegradable polymers and composites in biomedical applications: from catgut to tissue engineering. Part 1. Available systems and their properties. Int MaterRev 2004;49:261–273.

128. Bala I, Hariharan S, Kumar MN. PLGA nanoparticles in drug delivery: the state of the art. Crit Rev Ther Drug Carrier Syst 2004;21:387–422.

129. Mohanraj VJ, Chen Y. Nanoparticles - A review. Tropical Journal of Pharmaceutical Research 2006;5:561–573.

130. Hazer DB, Kılıçay E, Hazer B. Poly(3-hydroxyalkanoate)s: diversification and biomedical applications. A state of the art review. Mater Sci Eng C 2012;32:637–47.

131. Türesin F, Gürsel I, Hasirci V. Biodegradable polyhydroxyalkanoate implants for osteomyelitis therapy: in vitro antibiotic release. J Biomater Sci Polym Ed 2001;12:195–207.

132. Wang Y, Yin J, Chen GQ. Polyhydroxyalkanoates, challenges and opportunities. Curr Opin Biotechnol 2014;30C:59–65.

133. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 2012;161:505–522.

134. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev 2003;55:329–47.

135. Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE. Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release 2001;70:1–20.

136. Philip S, Keshavarz T, Roy I. Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J Chem Technol Biotechnol 2007;82:233–247.

137. Park H, Yang J, Seo S, Kim K, Suh J, Kim D, Haam S, Yoo KH. Multifunctional nanoparticles for photothermally controlled drug delivery and magnetic resonance imaging enhancement. Small 2008;4:192–196.

138. Kim J, Lee EJ, Lee SH, Yu JH, Lee JH, Park TG, Hyeon T. Designed fabrication of a multifunctional polymer nanomedical platform for simultaneous cancer-targeted imaging and magnetically guided drug delivery. Adv Mater 2008;20:478–483.

139. Lü JM, Wang X, Marin-Muller C, Wang H, Lin PH, Yao Q, Chen C. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn. 2009;9:325–341.

140. Bordes P, Pollet E, Avérous L. Nano-biocomposites: biodegradable polyester/nanoclay systems. Progress in Polymer Science 2009;34:125–155.

141. Avérous L, Pollet E. Green nano-biocomposites. L. Avérous and E. Pollet (eds.), 2012. Environmental Silicate Nano-Biocomposites, Green Energy and Technology, DOI: 10.1007/978-1-4471-4108-2_1. Springer-Verlag London.

142. Akaraonye E, Keshavarz T, Roy I. Production of polyhydroxyalkanoates: the future green materials of choice. Journal of Chemical Technology and Biotechnology 2010; 85:732–743.

Table 1. Summary of the developments on PHA nanobead protein functionalization for various applications.






PHA

Functionalization

GAP

Bacterial strain

Ref.

Diagnostics

PHB

Mouse interleukin 2 IL2/

myelin oligodendrocite glycoprotein MOG



PhaP phasin

PhaC


synthase

E. coli

18, 21

PHA

EFG/RFG/ Severe acute respiratory syndrome corona virus SARSCoV envelop protein

PHA

depolymerase



A. faecalis

22

PHB

Tuberculosis antigens, ESAT6, CFP10, and Rv3615c

PhaC

synthase


E. coli

23

PHB

Anti-β-galactosidase single-chain antibody variable fragment scFv

PhaC synthase

E. coli

24

Vaccines

PHB

M. tuberculosis antigen Ag85A-ESTAT-6

PhaC synthase

E. coli,

L. lactis

19,25, 26,27,28

PHB

Hepatitis C virus core antigen HCc

PhaC synthase

E. coli/

L. lactis

29

Drug delivery

PHBHHx

Mannosylated human α1-acid glycoprotein (hAGP)/human epidermal growth factor (hEGF)

PhaP phasin

In vitro

30

PHB

RGD

PhaC synthase

In vitro

31

PHB/ PHBHHx

Rhodamine B isothiocyanate RBITC

Non

In vitro

32

PHB

Rifampicin

Non

In vitro

33

PHBHHx

Triamcinolone Acetonide

Non

In vitro

34

PHB

Lomustine CCNU

Non

In vitro

35

PHBHHx

Heparzine-A

Non

In vitro

36

PHB

Diclofenac, dexamethasone

Non

In vitro

37

PHBHHx

Etoposide and attached folic acid

Non

In vitro

38

PHBHHx

Platelet-derived growth factor-BB (PDGF-BB)

Non

In vitro

39

Cell targeting

PHBHHx

Polyethylenimine coating

Non

In vitro

40

Imaging

PHB

GFP/ HcRed

PhaC synthase/

PhaP phasin



E. coli

41, 42

PHO

GFP

PhaF phasin

P. putida

8,9

PHB

Inorganic material binding peptide, antibody binding ZZ domain

PhaC synthase

E. coli

43

Insecticide

PHO

Cry1Ab

PhaF phasin

P. putida

16

Bioseparation




Immunoglobulin G (IgG) binding ZZ

domain of S. aureus Protein A



PhaC synthase

E. coli

13,44,45

PHB

ZZ

PhaC synthase

L. lactis

25

PHB

Streptavidin

PhaC synthase

E. coli

46

Protein purification

PHB

EGFP/Maltose binding protein MBP/

β-galactosidase (lacZ)-intein



PhaP phasin


R. eutropha

47

PHB

GFP,LacZ

PhaP phasin

R. eutropha

48

PHB

Intein self-cleaving affinity tag, EGFP, MBP, LacZ

PhaP phasin


E. coli

49

Enzymes

mclPHA

LacZ

PhaC synthase

P. aeruginosa

14

PHB

α-amylase variant (TermamylTM)

PhaC synthase

E. coli

50

PHB

Organophosphohydrolase

OpdA


PhaC synthase

E. coli

17

PHB

PhaA-PhaB

PhaC synthase

E. coli

51

Endotoxin removal

PHB

Lipopolysaccharide binding protein

PhaP phasin

In vitro

52

Yüklə 250,95 Kb.

Dostları ilə paylaş:
1   2   3




Verilənlər bazası müəlliflik hüququ ilə müdafiə olunur ©muhaz.org 2024
rəhbərliyinə müraciət

gir | qeydiyyatdan keç
    Ana səhifə


yükləyin